Laser-activated Bioprobes with High Photothermal Conversion

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Biological and Medical Applications of Materials and Interfaces

Laser-activated Bioprobes with High Photothermal Conversion Efficiency for Sensitive Photoacoustic/ Ultrasound Imaging and Photothermal Sensing Hongrui Zhu, Dui Qin, Youshen Wu, Bowen Jing, Jiajun Liu, David Hazlewood, Hongmei (Clara) Zhang, Yi Feng, Xinmai Yang, Mingxi Wan, and Daocheng Wu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b08190 • Publication Date (Web): 13 Aug 2018 Downloaded from http://pubs.acs.org on August 13, 2018

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ACS Applied Materials & Interfaces

Laser-activated Bioprobes with High Photothermal Conversion Efficiency for Sensitive Photoacoustic/ Ultrasound Imaging and Photothermal Sensing Hongrui Zhua#, Dui Qina#, Youshen Wub, Bowen Jinga, Jiajun Liua, David Hazlewoodc, Hongmei Zhanga, Yi Fenga, Xinmai Yangc, Mingxi Wana*, Daocheng Wua* a

Key Laboratory of Biomedical Information Engineering of Education Ministry, School of Life

Science and Technology, Xi'an Jiaotong University, Xi'an 710049, PR China b

Department of Chemistry, School of Science, Xi'an Jiaotong University, Xi'an 710049, PR

China c

Bioengineering Research Center and Department of Mechanical Engineering, University of

Kansas, Lawrence, KS, USA #

These authors contributed equally to this work.

*

Corresponding authors.

Email address: [email protected] (D. Wu), [email protected] (M. Wan) Keywords: photothermal conversion; bioprobes; perfluorocarbon; nanodroplets; sensor

ACS Paragon Plus Environment

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ABSTRACT

Laser-activated bioprobes with high photothermal conversion efficiency (IRPDA@PFH NDs) based on biocompatible IR-780 doped polydopamine perfluorocarbon nanodroplets were developed. When protected by gelatin microspheres, their near-spherical morphologies can be easily observed with transmission electron microscope. Doping IR-780 (3% w/w of added dopamine hydrochloride) could significantly enhance near-infrared (NIR) absorption and photothermal conversion efficiency to 57.7%. The enhanced NIR absorption and non-radiative relaxation are preferred to stronger photoacoustic (PA) signals and higher PA imaging definition, ultrasound (US) signals also increase more than 2.5 times due to easier phase change of nanodroplets. These bioprobes had sensitive PA/US imaging capability with highly effective substitute utilizations, in which polydopamine was either used as PA contrast or photothermal agent. Perfluorocarbon could be used as US contrast agent and temperature indicator. More importantly, the gray value increments of US increase with temperature in a general range from 35 °C to 55 °C. An approximate linearity of gray value increments variation in the optimized photothermal therapy (PTT) range from 35 °C to 50 °C could be used for the temperature monitoring and control of PTT, heated regions, as well as the extent of photothermal heating, can be visualized by US imaging. These findings indicate their great potential for biosensing and PTT monitoring.

1. INTRODUCTION Photoacoustic (PA) imaging provides high contrast and ideal spatial resolution in deep biological tissue and is a powerful diagnostic tool.1 However, ultrasound (US) imaging still plays an important role in biomedical imaging because of its high safety, low cost, portability, and high


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spatial and temporal resolution.2 These advantages make ultrasound imaging an ideal candidate for biomedical process monitoring. A contrast agent is needed to improve the imaging quality and low contrast of US imaging, and microbubbles are usually used as US contrast agent, for the dramatically increased contrast due to the mismatched ultrasound impedance between microbubbles and host tissue.3 Perfluorocarbon nanodroplets are superior to traditional microbubbles US contrast agents in many aspects,4 Marshalek et al. prepared folate receptortargeted phase-change contrast agents composed of perfluorocarbon cores encapsulated by lipid membranes with target ligands attached. Their feasibility of intracellular delivery and activation were

demonstrated.5

Laser-activated

phase-change

nanodroplets,

which

consist

of

perfluorocarbon and light absorber, have recently emerged, and they connect PA imaging with US imaging.6-8 PA signals are provided by the thermal expansion of their absorbers and the vaporization of perfluorocarbon droplets (microbubbles generation). In addition, increased echogenicity of the resulting perfluorocarbon microbubbles makes them excellent US contrast agents. These previously reported laser-activated phase-change droplets showed the capabilities of PA/US imaging, and some of them can serve as photothermal therapy (PTT) agents. For example, Tang et al. developed an organic semiconducting photoacoustic nanodroplet composed of low-boiling-point perfluorocarbon, perylene diimide and photosensitizer. This photoacoustic nanodroplet exhibited dual-modal PA/US imaging, photothermal therapy and oxygen released from it enhanced photodynamic therapy.6 Li and colleagues designed agents composed of mesoporous silica filled with gold nanorod embedded perfluorocarbon for PA/US bimodal imaging-guided photothermal therapy, showing great potential for cancer therapy.8 However, their unsatisfying absorption and photothermal conversion efficiency caused relatively inferior PA imaging quality, and not to mention to acquire both PA and US images of high quality due to


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the lower photothermal conversion efficiency of their absorbers and utilization of the systems. These reports conducted US imaging and PTT separately,9 and their PTT effect could not be sensed effectively similar to most nanoparticle-based PTT studies. For example, the commonly used infrared imaging sensing method could only sense the surface temperature of tissues, and an invasive implanted electrode could only measure a single-point temperature.10 As a kind of phase-change material, perfluorocarbon possesses temperature responsiveness and can be changed from liquid to gas after heating. It has the potential to be used for temperature sensing, although no perfluorocarbon-based PTT sensors have been reported.

Scheme 1. Illustration of design principles of IRPDA@PFH NDs. PFH NDs were emulsified from bulk PFH in Tris solution via ultrasonication. Polydopamine started coating on PFH NDs and IR-780 was doped into it. Decreased fluorescence and enhanced NIR absorption are preferred to higher photothermal conversion ability. IRPDA@PFH NDs can be used as PA/US contrast agents and PTT sensors. High absorption and photothermal conversion efficiency are preferred when enhancing the non-radiative relaxation (fluorescence quenching) of chromophore.11, 12 As a fluorescence dye


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with suitable excitation and emission wavelength match the commonly used NIR laser, IR-780 has been widely used as PA imaging and PTT agent. It also shows better properties than other similar NIR dyes (e.g. indocyanine green) with higher and more stable fluorescence intensity.13 Polydopamine is a melanin-like polymer and a promising NIR absorber for photothermal conversion, and it has been widely used in the biomedical field. 14 It is worth mentioning that polydopamine is also a friendly coating material, and many studies have proved that it could improve the safety and stability of biomaterials.15,

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In addition, polydopamine has strong

fluorescence quenching capability, which increases non-radiative transition. Inspired by these, a novel strategy was developed to design the laser-activated bioprobes with high photothermal conversion efficiency based on IR-780 doped polydopamine perfluorohexane nanodroplets (IRPDA@PFH NDs), in which IR-780 (3% w/w of added dopamine hydrochloride) was doped in the polydopamine shell (Scheme 1). The doping of IR-780 greatly increased the light absorption and the fluorescence quenching of IR-780 could vanish its light emission. Thus, the photothermal conversion efficiency of the system was greatly enhanced. The experimental results agree with our assumption, that is, the photothermal conversion efficiency of the system is greatly enhanced to 57.7%, which is higher than that of pure polydopamine and IR-780. These bioprobes with satisfying photothermal activity and phase-change capability have better PA/US bimodal imaging than that of previous reports.8, 17-20 IRPDA@PFH NDs are also highly efficient bioprobes with high substitutes and function utilization. Polydopamine shell either served as a carrier for IR-780 and perfluorocarbon or photothermal conversion agent. IR-780 not only served as a NIR absorber and photothermal conversion enhancer, but also generated PA signals. Perfluorocarbon was used as US contrast agent and temperature indicator. Moreover, US signals have a certain relationship with photothermal temperatures and are suitable for sensing their PTT


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effect. Heated regions, as well as the extent of photothermal heating, can be visualized by US imaging, indicating their great potential for biosensing and PTT monitoring. Doping IR-780 into polydopamine is an important method to develop highly efficient PA and photothermal agent, indicating its important significance for the bioprobe designs. 2. EXPERIMENTAL SECTION 2.1. Materials. Perfluorohexane (PFH) was purchased from Apollo Scientific Co. Ltd (UK). FS-63 was purchased from DuPont Co. Ltd (USA). Tris was purchased from MP Biomedicals (France). IR-780 was purchased from Sigma–Aldrich (USA). Dopamine hydrochloride was purchased from Aladdin Co. Ltd. (China). The other reagents were purchased from China Tianli chemical reagent Co. Ltd. All reagents were of analytical grade and used without further purification. HeLa and PC-3 cells were purchased from American Type Culture Collection. Cell culture supplies were purchased from Corning Incorporated and GE Healthcare HyClone™ Cell Culture. Female nude BALB/c mice were purchased from the animal experimental center of Xi’an Jiaotong University. The animal experiments were approved by the ethics committee of Xi'an Jiaotong University. 2.2. Preparation and characterization of IRPDA@PFH NDs. Typically, 100 µL of PFH and 40 µL FS-63 were added into 4 mL of 50 mM Tris solution. The mixture was placed under ultrasonication using a conical tip sonicator instrument (JYD-150, Zhisun equipment, China), and the US program was set to 3 seconds on/3 seconds off for 10 cycles with 20% power in an ice water bath. Then, the mixture turned into a milky nanoemulsion. 100 µL of 5 mM IR-780 dimethyl sulfoxide solution and 1 mL of 10 mg/mL dopamine hydrochloride solution were added


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into the nanoemulsion. The mixture should be gently blended continuously before adding approximately 200 µL CuSO4 (5 mM)/H2O2 (19.6 mM) stock solution. When all the reagents were added, the mixture was kept rotating in a rotator for more than 1 h. To remove free ions and IR-780, the mixture was dialyzed in a dialysis bag (5 KD) for 24 h, and prepared IRPDA@PFH NDs were stored at 4 °C for further use. The amount of IR-780 in IRPDA@PFH NDs was determined by standard curve made according to UV-vis spectra method, and the doping efficiency was determined by21

Doping efficiency =

() － () × 100%

()

Morphology and size were characterized by transmission electron microscopy (TEM), (H7650, Hitachi, Japan) after the samples were dropped on a copper grid coated with ultrathin carbon support film for 10 min. IRPDA@PFH NDs samples were protected by gelatin microspheres for TEM characterization, which were prepared by a modified glutaraldehyde cross method.22 The Fourier transform infrared (FTIR) spectroscopy was recorded by FTIR instrument (VERTEX70, Bruker Corporation, Germany), and the samples for testing were dried in a drying oven after centrifugation. The UV-visible spectra and absorption value at 808 nm (A808) were recorded by UV-visible spectrophotometer (T6, Purkinje general, China). The fluorescent emission spectra were obtained by fluorescence spectrometer (Fluoromax-4, Horiba, Japan). 2.3. Cell experiments. The viability and proliferation of HeLa cervical cancer cells were evaluated by methyl thiazolyl tetrazolium (MTT) assay. HeLa cervical cancer cells were cultured at 37 °C with 5% CO2 for 24 h. Subsequently, the cultured medium was removed, and the cells were incubated with IRPDA@PFH NDs for another 24 h and washed using the medium twice. Then, 100 µL of the new culture medium that contains


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MTT reagent (10%) was added to each well of the 96-well assay plate and incubated for 4 h. Then, the medium was removed, and the purple formazan product was dissolved using DMSO for 15 min. The optical absorption at 490 nm was measured by a multimode reader (SpectraMax M2e, Molecular Devices, USA). The photothermal effect of IRPDA@PFH NDs for cells was evaluated. HeLa cells were incubated with IRPDA@PFH NDs (200 ug/mL) in six-well plates at 37 °C for 30 min, and then a NIR laser (808 nm, 1.0 W cm-2) irradiated onto them at the same spot for 5 min. Subsequently, the cells were stained with calcein acetoxymethyl (AM) ester and propodium iodide (PI). Photothermal cytotoxicity of IRPDA@PFH NDs with different concentrations was also quantified. The cells were exposed to a 1.0 W cm−2 808 nm laser for 5 min and then incubated for 24 h. The results of photothermal effect for cells were quantified by MTT assay. 2.4. Measurement of photothermal activity. 1.5 mL of IRPDA@PFH NDs with different concentrations (25–200 ug/mL) were suspended in a fixed 1.5 mL centrifuge tube and were exposed to an 808 nm laser with different powers (1.0–2.0 W cm−2) for 10 min. Their temperatures were recorded every 30 seconds for 20 min (including the temperature-fall period) using a thermocouple. Double distilled water and PFH nanodroplets without PDA coating (PFH NDs) and PDA@PFH NDs without doping IR780 served as blank controls. Infrared thermal images of samples were recorded using an infrared thermal imaging camera (FLIR, USA). Balb/c male nude mice (18–22g, approximately 6 weeks old) that bear tumor were divided into PBS, IRPDA@PFH NDs, and their corresponding groups with NIR laser irradiation at a power density of 1.5 W cm−2 for 5 min, the infrared images were captured every 30 s. Tumor


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sections for hematoxylin and eosin (H&E) staining were prepared 2 days after laser irradiation to evaluate the PTT activity of IRPDA@PFH NDs. 2.5. PA and US imaging. PA imaging: After anesthetizing tumor-bearing nude mice, 50 µL of IRPDA@PFH NDs (1.6 mg/mL) were intratumorally injected, PA signals were collected using a home-built PA imaging system. A 760-nm Q-switched Nd:YAG laser pumped an optical parametric oscillator laser with 6 ns pulses at a repetition rate of 10 Hz (20 mJ cm−2), and a 5 MHz transducer collected the PA signals. US imaging in phantom: A phantom made of 2% agarose and IRPDA@PFH NDs was placed into a water tank, and a 10 MHz array transducer connected with an ultrasonic imaging device (SonixTouch, Ultrasonix, Richmond, BC, Canada) was used to detect the phantom vertically. US images before and after 808-nm NIR laser irradiation (1.0 W cm−2) were acquired, and a shape of four capital letters (“XJTU”) was set as the laser path. US bio-imaging: Approximately 500 µl of IRPDA@PFH NDs were injected into a chicken tissue, and a syringe needle was left as a position mark. Before irradiation of an 808 nm NIR laser, an US image of the tissue was acquired using an US system to establish a background as control. After 808 nm NIR laser irradiation, another US image was acquired as the experiment data. For in vivo imaging, PC-3 tumor-bearing nude mice model was obtained. A total of 200 µL of IRPDA@PFH NDs (1.6 mg/mL) was intratumorally injected, and US images were acquired before and after 808 nm laser irradiation for 10 min. US gray values of tumors were measured by Image-Pro Plus software, and temperatures were measured using an infrared thermal imaging camera to investigate the relationship between US values and photothermal temperatures.


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3. RESULTS AND DISCUSSION 3.1. Preparation and characterization. The preparation process was illustrated in Scheme 1. We adapted coating method to connect the absorber with the perfluorocarbon nanodroplet, which is more convenient than perfluorinating and embedding absorber into perfluorocarbon nanodroplet.17, 23-25 . Polydopamine is formed by dopamine self-polymerization; it can virtually coat onto all materials in weak alkaline media, regardless of their chemistry reactions.26-28 Polydopamine has more advantages, such as easier coating process and better biocompatibility, than those of coating materials for other laser-activated nanodroplets. Although polydopamine polymerizing is a time-consuming progress (general method consumes approximately 12 hours), a highly effective method was developed to realize rapid polydopamine coating on PFH nanodroplets with the help of reactive oxygen species produced by CuSO4/H2O2

29, 30

. For

quenching fluorescence and enhancing non-radiative relaxation of IR-780, small amount of IR780 (less than 0.2% w/w of IRPDA@PFH NDs or 3% w/w of added dopamine hydrochloride) was added into the mixture and doped in the shell during the polydopamine-coating process. The doping efficiency of IR-780 was calculated to be 75%. Such a small amount of IR-780 greatly improves their photothermal conversion efficiency. Clear and representative images with normal TEM are difficult to acquire because the high vacuum of electron microscope system causes perfluorocarbon change phase from liquid to gas. Thus, previous reports of perfluorocarbon nanodroplets did not show TEM images;31, 32 other researchers used cryo-TEM to deal with these nanodroplets,18, 33 but their morphologies were not the same as that in the solution and the technical requirement and cost for cryo-TEM were high. In the current study, IRPDA@PFH NDs were embedded into transparent gelatin microspheres, which protected samples from vaporization. The morphology and size clearly showed its


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similarity with the solution. Figure 1A shows that IRPDA@PFH NDs are embedded in gelatin microspheres, and each gelatin microsphere contains one to four IRPDA@PFH NDs with an average size of 120 nm. The inset of Figure 1A shows a magnified image of near-spherical IRPDA@PFH NDs with a core-shell structure. The FTIR results of IRPDA (shell of IRPDA@PFH NDs) in Figure 1B have the characteristic peaks of polydopamine that are in agreement with the references.34,

35

Weak absorption peaks of IRPDA at 1300–1000cm−1

attributes to the C–N stretching vibration absorption of aromatic rings and indicates that IR-780 is embedded in the polydopamine shell. Xu et al. reported that oxygen radical produced by CuSO4/H2O2 can accelerate polydopamine coating on bulk materials only within 1 hour, and we confirm it can also accelerate polydopamine coating on PFH nanodroplets (PFH NDs). The polydopamine polymerization processes reflected by apparent color and absorption values at 808 nm are showed in Figure 1C and Figure 1D. The darkness degree and absorbance values at 808 nm of PDA@PFH NDs with CuSO4/H2O2 increased faster and higher than the group without CuSO4/H2O2. The results indicate that CuSO4/H2O2 can accelerate polydopamine coating and reduce the coating time to 1 hour. Figure 1E shows the UV-visible spectroscopies of various materials. PDA@PFH NDs had a wide absorption range (blue line), and the addition of CuSO4/H2O2 slightly enhanced their absorption (red line). When doped into PDA shell, the fluorescent intensity of IR-780 decreased sharply (Figure S1) due to the quenching effect of polydopamine. The decreased fluorescence prefers to enhance non-radiative relaxation, which can disregard its light emission and photothermal conversion efficiency was significantly increased due to energy transformation. Both polydopamine and IR-780 have strong absorption, and their complex would have higher absorption than polydopamine and IR-780 respectively. After IR-780 and polydopamine interacted, the environment for molecular vibrations related to


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absorption was changed. Free IR-780 had weak light scatter ability, while nanoparticles it was embedded in showed less light transmittance and higher absorption. Moreover, polydopamine reduced the fluorescence emission because of the quench effect, and thus the absorption would be enhanced. Thus, the combination of polydopamine and IR-780 showed higher absorption than polydopamine and IR-780. Therefore, the absorption of IRPDA@PFH NDs (orange line) at 808 nm was 1.8 times higher than that of PDA@PFH NDs (blue line) and 11.7 times higher than that of free IR-780 (green line). As a result, PFH NDs acquired excellent NIR absorption when coated with polydopamine and small amount of IR-780. Such an enhanced NIR absorption is greatly helpful to acquire strong signals and good sensitivity for PA imaging. The absorption at 808 nm of IRPDA@PFH NDs with and without CuSO4/H2O2 showed few differences, indicating that CuSO4/H2O2 does not affect the absorption of IRPDA@PFH NDs. Light might induce IR780 decomposition because of its poor photostability.36 IR-780 can acquire high photostability when incorporated into the shell of PDA@PFH NDs, which protects it from photobleaching. Figure 1F compares the photostability of free IR-780 (83µM) and IRPDA@PFH NDs with the same amount of IR-780 exposed to sunlight for 24 h. Free IR-780 changed their color from dark green to pale brown, and the absorption at 808 nm decreased from 0.114 to 0.004, whereas IRPDA@PFH NDs still maintained their appearance and absorption value at 808 nm slightly changed. The possible mechanism might be polydopamine shells increased the intermolecular distance of free IR-780 and π–π stacking was avoided.37 3.2. Enhanced photothermal conversion efficiency. There were not many reported laseractivated nanodroplets with high photothermal conversion capability, and none of them was used as bioprobes or PTT sensor. Results in Figure 2A show the temperature of 200 µg/mL IRPDA@PFH NDs at a laser power density of 1.0 W cm-2 increases by 48.8 °C in 600 s,


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whereas the temperature of pure water and PFH NDs at the same concentration increases less than 10 °C, indicating that IRPDA@PFH NDs have excellent photothermal conversion capability (efficiency is 57.7% and detailed calculation process of photothermal conversion efficiency was written in Supporting Information, Figure S8).

Figure 1. The characterization of IRPDA@PFH NDs. (A) A TEM image of PFH NDs in gelatin microspheres (scale bar: 500nm), the inset is a magnified image of IRPDA@PFH NDs (scale bar: 100nm). (B)The FTIR spectra of PDA, PDA+CuSO4/H2O2 and IRPDA (C) Polymerization process of PDA@PFH NDs with and without CuSO4/H2O2. (D)Absorbance value change of


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PDA@PFH NDs with and without CuSO4/H2O2 at 808nm. (E)UV-visible spectra of PDA@PFH NDs, IR-780, PDA@PFH NDs+CuSO4/H2O2, IRPDA@PFH NDs and IRPDA@PFH NDs (without CuSO4/H2O2). (F) Photostability test of free IR-780 and IRPDA@PFH NDs. Compared with pure water, PFH NDs and PDA@PFH NDs, IRPDA@PFH NDs showed the highest temperature in infrared thermal imaging photos (Figure 2C). Such photothermal capability is stronger than other PA or PTT agents, such as gold nanorods (efficiency 39%),38 polydopamine nanoparticles (efficiency 40%),34 and free IR-780 (efficiency 17%) (Supporting Information, Figure S9, and PDA@PFH NDs (efficiency 46.4%) (Supporting Information, Figure S10). Fluorescence quenching and non-radiative relaxation are helpful to the energy transfer from light to heat after IR-780 doping. This strategy is helpful for the design of highly efficient PA and PTT agents. Under the same conditions, the temperature of polydopamine (with a greater laser power of 2.0 W cm−2) increased less than 35 °C,34 and the temperature of free IR780 increased less than 20 °C.21 Temperature increasing profiles at different concentrations (Figure 2B) and power densities (Figure S2) indicate that IRPDA@PFH NDs can be further heated with higher concentration and laser power density. Figure S3 demonstrates that if IRPDA@PFH ND quantity is sufficient, then they can be heated for several cycles. This phenomenon indicates that IRPDA@PFH NDs can be heated by laser efficiently and have great potential to be used as PA imaging agents. In addition, microbubbles generated from phasechanged perfluorocarbon nanodroplets are widely used as US contrast agents.39 Such a high photothermal conversion efficiency easily proves the phase change of nanodroplets and thus increases US sensing sensitivity. Laser-induced bubble-generating process of IRPDA@PFH NDs was also investigated. After laser irradiated for 10 s, a few small bubbles began to appear, and the appearance of bubbles increased with time. Some bubbles fused together and expanded, and


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it was easy to find many bigger bubbles came into view at 35 s (Figure 2D). These results suggest that IRPDA@PFH NDs are suitable to serve as highly sensitive US contrast agents.

Figure 2. Photothermal property of IRPDA@PFH NDs. (A) The temperature profile of water, PFH NDs and IRPDA@PFH NDs irradiated with a laser, followed by natural cooling after laser turned off. (B) The photothermal heating curves of IRPDA@PFH NDs with different concentrations under laser irradiation. (C) Infrared thermal images of water, PFH NDs


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IRPDA@PFH NDs and PDA@PFH NDs irradiated with laser. (D) Optical microscopy images of IRPDA@PFH NDs phase change under 808nm laser irradiation (scale bar: 200µm). 3.3. Photothermal effect evaluation. The cell photothermal effect of IRPDA@PFH NDs was evaluated. The results in Figure 3B show that IRPDA@PFH NDs had no detectable cytotoxicity on HeLa cells with a high concentration of 200 ug/mL, and only NIR laser (0.00 ug/mL) cannot affect cells obviously. In contrast, when HeLa cells were incubated with IRPDA@PFH NDs (200 ug/mL) and irradiated with laser (808 nm, 1.0 W cm−2) for 5 min, their cell viability significantly decreased. When cells treated with IRPDA@PFH NDs (200 ug/mL) were stained with calcein-AM and PI, a distinct line between the areas with and without laser irradiation could be observed. Cells irradiated with laser were dead (red), whereas cells without laser irradiation were live (green) (Figure 3A). While all cells only treated with laser were all live (green) (Figure S4). These results suggest that IRPDA@PFH NDs have excellent biocompatibility and low toxicity and can kill cancer cells effectively when combined with NIR laser. Infrared thermal imaging photos in Figure 3D& Figure S5 showed that IRPDA@PFH NDs could be laser heated after intra tumor injection. The temperature increased from 33.4 °C to 56.3 °C when irradiated with an 808nm laser at 1.5 W cm−2 in 5 minutes, and such a temperature was high enough to kill tumors efficiently. H&E staining images were shown in Figure 3C. Only necrotic and apoptotic tumor cells could be observed in the images of tumors treated with IRPDA@PFH NDs+laser. In addition, caves were left due to the phase change of nanodroplets, whereas images of tumor cells treated with PBS, PBS+laser, and IRPDA@PFH NDs did not show obvious damage. These results indicate that IRPDA@PFH NDs have excellent photothermal activity in tumor tissue and can be used as promising PTT agents.


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Figure 3. Test of photothermal therapy ability (A) Images of calcein-AM and PI stained Hela cells after laser irradiation. (B) Cell viability of cells incubated with IRPDA@PFH NDs of different concentrations and corresponding groups with laser irradiation. (C) Images of H&E stained tumor sections from tumor bearing mice receiving treatments. (D) Infrared thermal images of tumor bearing mice before and after laser irradiated for 5 min. 3.4. PA/US imaging and PTT sensing. Our bioprobes show higher NIR absorption and non-radiative quantum yield than other laser-activated nanodroplets and many PA contrast agents, which are two important parameters for PA imaging.40 PA imaging was conducted on tumors after the injection of IRPDA@PFH NDs. Figure 4A shows that the region of interest has very strong PA signals and two distinct tumors can be identified. The definition and contrast of PA images are superior to other laser-activated phase-change nanodroplets17, 41 because of the outstanding NIR absorption and enhanced non-radiative relaxation after doping a small amount of IR-780. These results indicate that IRPDA@PFH NDs can be used as excellent


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PA agents with high performance. To verify their US imaging capability, US images of a phantom before and after 808-nm NIR laser irradiation was acquired, and four capital letters (“XJTU”) were set as the laser path. The agarose phantom mixed with IRPDA@PFH NDs served as a piece of “paper,” the 808-nm NIR laser played the role of a “pen,” and an US transducer served as the detector. Figure 4B-i and ii show the US images before and after laser beam wrote, and “XJTU” can be read out on the screen. In addition, IRPDA@PFH NDs show the potential of US imaging in biological tissue when injected into a chicken tissue (the needle was left as a position marker). Figure 4B-iii and iv show that the US images in the region of interest are brighter after laser irradiation. The results show that IRPDA@PFH NDs can serve as efficient US contrast agents for biological tissue. Figure 4C shows a clear tumor image, and regions brighten after laser irradiation (808 nm 1.5 W cm−2). US signals (gray values) of two images in Figure 4C were also measured and drawn as a column chart (Figure 4D). The US signals after laser irradiation were approximately 2.5 times stronger than before, indicating that US signals enhanced after photothermal conversion. High photothermal conversion effect resulted in greater enhancement than many previous reports.19 For example, the US enhancement of a recent report8 was less than 2 times after laser irradiation. These results indicate that IRPDA@PFH NDs are excellent US contrast agents. Photothermally heated regions can be judged based on US images. The area of the blue dashed lines encircled in Figure 4C showed few US signals before PTT. After laser irradiation for 10 min, regions that received effective PTT (red dashed lines circled) turned bright and white, whereas the areas that were not directly heated by PTT (blue


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dashed lines circled) remained black. Moreover, temperature increase can be reflected by the intensity of US signals. Ultrasound images and infrared thermal photos were showed in Figure S6, the variations of gray value increments and temperature were plotted in Figure 4E, and their values increased with time. To explore the relationship between US signals and temperatures, the changing curves of gray value increments were plotted with temperatures roughly (Figure 4F). Figure 4F shows that the gray value increments increase with temperatures in a general range from 35 °C to 55 °C. The trend of gray value increments variation with temperature from 35 °C to 50 °C is approximate linearity (Y=11943X-391677, R2=0.94156, Supporting Information, Figure S7 ), and such a temperature range is an optimized choice for PTT. When temperature was higher than 60 °C, which exceeded the boiling point of PFH, the curve tended to be flat because most IRPDA@PFH NDs had been vaporized. The results indicate that temperature variation in PTT can be reflected by US signals with the aid of our bioprobes.


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Figure 4. PA/US imaging and PTT sensing results after IRPDA@PFH NDs injection. (A) Photoacoustic images of tumors injected with IRPDA@PFH NDs. (B) i, ii showed US images of an agarose phantom mixed with IRPDA@PFH NDs before and after laser writing on it. iii, iv showed the US images of a chicken tissue before and after laser irradiation (C) Ultrasound


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images of tumor before and after photothermal therapy. Blue circle represented regions didn’t receive effective therapy, red circle showed that area received effective therapy. (D) A comparing between Gray values from two images in (C). (E) The gray value increments and temperature of tumors changed with time (mean±SD, n=3). (F) Changing curves of gray value increments with temperatures (mean±SD, n=3). 4. CONCLUSION Laser-activated bioprobes with high photothermal conversion efficiency were developed based on IR-780 doped polydopamine perfluorocarbon nanodroplets, in which IR-780 (3% w/w of added dopamine hydrochloride) was doped in the polydopamine shell. The enhanced optical energy absorbed by IR-780 was transferred to heat, which greatly increased photothermal conversion capability, because of fluorescence quenching and non-radiative relaxation. These bioprobes had sensitive PA/US imaging capability with highly effective substitute utilization, in which all constitutes had two or more functions. Moreover, the relationship between US imaging and photothermal temperature, which would be helpful for biosensing and PTT monitoring, was discovered. ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge on the ACS Publications website. Fluorescent spectra, temperature profile and variations of IRPDA@PFH NDs, infrared thermal images and ultrasound images of tumor bearing mice, linearity of gray value increments increased with temperature and the calculation of photothermal conversion efficiency.


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ACKNOWLEDGMENT The authors thank Lingze Zhang, Yiming Liu and Peng Tang for assistance with the animal experiments. This work was sponsored in part by National Natural Science Foundation of China (81471771 and 11674262) , the grants of National Key Research and Development Program of China (NO. 2016YFC0100701). REFERENCES 1.Wang, L. V.; Hu, S., Photoacoustic Tomography: In Vivo Imaging from Organelles to Organs. Science 2012, 335, 1458-1462. 2.Perera Reshani, H.; Hernandez, C.; Zhou, H.; Kota, P.; Burke, A.; Exner Agata, A., Ultrasound Imaging Beyond the Vasculature with New Generation Contrast Agents. Wiley Interdiscip. Rev.Nanomed. Nanobiotechnol. 2015, 7, 593-608. 3.Nakatsuka Matthew, A.; Mattrey Robert, F.; Esener Sadik, C.; Cha Jennifer, N.; Goodwin Andrew, P., Aptamer-Crosslinked Microbubbles: Smart Contrast Agents for Thrombin-Activated Ultrasound Imaging. Adv. Mater. 2012, 24, 6010-6016. 4.Xu, X.; Song, R.; He, M.; Peng, C.; Yu, M.; Hou, Y.; Qiu, H.; Zou, R.; Yao, S., Microfluidic Production of Nanoscale Perfluorocarbon Droplets as Liquid Contrast Agents for Ultrasound Imaging. Lab on a Chip 2017, 17, 3504-3513. 5.Marshalek, J. P.; Sheeran, P. S.; Ingram, P.; Dayton, P. A.; Witte, R. S.; Matsunaga, T. O., Intracellular Delivery and Ultrasonic Activation of Folate Receptor-Targeted Phase-Change Contrast Agents in Breast Cancer Cells in Vitro. J. Control. Release 2016, 243, 69-77. 6.Tang, W.; Yang, Z.; Wang, S.; Wang, Z.; Song, J.; Yu, G.; Fan, W.; Dai, Y.; Wang, J.; Shan, L.; Niu, G.; Fan, Q.; Chen, X., Organic Semiconducting Photoacoustic Nanodroplets for Laser-


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Table of Contents Graphic and Synopsis

Laser-activated bioprobes with high photothermal conversion efficiency were developed based on IR-780 doped polydopamine perfluorocarbon nanodroplets. Small amount of IR-780 greatly enhanced NIR absorption and photothermal conversion capacity, PA/US imaging and PTT sensing.


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Scheme 1 56x36mm (300 x 300 DPI)



Figure 1 133x151mm (300 x 300 DPI)


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Figure 2 149x229mm (300 x 300 DPI)



Figure 3 89x57mm (300 x 300 DPI)


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Figure 4 171x191mm (300 x 300 DPI)



Figure S1 70x46mm (300 x 300 DPI)


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Laser-activated Bioprobes with High Photothermal Conversion

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